One (1) of the key goals in cardiac regeneration is to establish new myocardium that is as close as possible to normal myocardium, both structurally and functionally. Normal mammalian myocardium has an intricate network of capillaries, with each myocyte next to at least 1 capillary, and endothelial-cardiomyocyte signaling in myocardium regulates not only development of the myocardium, but also myocyte function in adult myocardium. Ultimately, achieving the goal of regenerating myocardium will require not only restoration of viable cardiomyocytes and endothelial cells, but promoting the intricate structural relationship of myocytes and the microvasculature. The primary significance of these experiments is to advance understanding of the interactions of cardiomyocytes with the microvasculature to improve our capabilities of establishing normally-functioning regenerated myocardium. In the preliminary studies of this proposal, we present data indicating that cardiomyocytes and endothelial cells retain the ability to assemble in a manner analogous to the pericyte-endothelial assembly and stabilization steps of vasculogenesis. Similarly, our preliminary data show that in 3D culture, differentiated endothelial cells form capillary structures, and cardiomyocytes attach to the outside of these capillary structures, allowing the endothelial cells to prevent cardiomyocyte apoptosis. Here we propose 3 Aims investigating how cardiomyocyte-endothelial interactions may play a crucial role in re-establishing myocardial architecture. Aim 1. To determine mechanisms used by endothelial cells to improve viability of nearby or adjacent cardiomyocytes. Aim 2. To demonstrate in vivo that adjacent endothelial cells improve viability, growth, and maturation of cardiomyocytes, and to determine the mechanisms for these benefits. Aim 3. To determine if endothelial cells can promote the development of new, alpha-Myosin Heavy Chain-positive mature cardiomyocytes from alpha-Myosin Heavy Chain-negative myocyte precursor cells, using genetic fate-mapping in mice.